The presence of nucleic acids in a sample may indicate that a source from which the sample is taken has a disease, disorder or abnormal physical state. Certain diagnostics determine whether nucleic acids are present in a sample. These diagnostics invariably require amplification of nucleic acids because of the copy number problem. In a virus or cell, for example, there is usually a single copy of a particular gene. Without amplification of specific nucleic acids of the gene, it is often difficult to detect the presence of the nucleic acids.
There are a number of processes for amplifying nucleic acids. Two such processes are those described in U.S. Pat. No. 5,130,238 (Malek et al.) and U.S. Ser. No. 07/211,384 (Malek et al.), U.S. Pat. No. 5,409,818. Malek's amplification processes require less participation and fewer manipulations by the user. The amplification cycle takes place at a relatively constant ambient temperature and without the serial addition of reagents. The template nucleic acid sequence generates more than one product from one substrate in each cycle of the amplification process. The amplification processes use either DNA or RNA as a starting template.
Where single or double-stranded DNA is the starting template, Malek's processes require thermal or alkali denaturation before the amplification cycle.
Where single-stranded DNA is a starting template, a first primer hybridizes to the DNA. Then, a DNA-directed DNA polymerase makes a double-stranded DNA product. The product of the polymerization then undergoes either thermal or alkali denaturation before the single-stranded DNA enters the cycle.
Where double-stranded DNA is a starting template, the double-stranded DNA first undergoes either thermal or alkali denaturation. The first primer then hybridizes to one of the single strands of DNA. Then, a DNA-directed DNA polymerase makes a double-stranded DNA product. The product of the polymerization then undergoes a second thermal or alkali denaturation before the single-stranded DNA enters the cycle.
Thermal denaturation is problematic because entry into the amplification cycle is not isothermal. Alkali denaturation is problematic because it requires participation and manipulations by a user of the amplification process. Thermal denaturation may be used not only in Malek's processes, but in other amplification processes (for example, PCR and LCR).
Thus, a need exists for improvements to nucleic acid amplification to (1) decrease the number of steps involved in the process, (2) decrease the participation and manipulations by a user, (3) eliminate the heating steps involved in entering any amplification cycle, and (4) reduce the temperatures at which any heating takes place.
In this application, the phrase "specific nucleic acid sequence" means a sequence of single-stranded or double-stranded nucleic acids or a sequence complementary to such sequence which one wishes to amplify. "DNA-directed RNA polymerase" means such polymerase of a quality suitable for use in molecular biological reactions. One "unit" of a first RNA polymerase means the amount of RNA polymerase which catalyzes the incorporation of one nanomole of radiolabelled ribonucleoside triphosphate into an RNA first template in 10 minutes at 37.degree. C.